1. Field of the Invention
The present invention relates to a solid substrate for a biochip having a uniform spacing of predetermined functional groups and a biochip using the same. More particularly, the present invention relates to a rotaxane compound, a rotaxane compound-bonded solid substrate, and a biochip using the solid substrate.
2. Description of the Related Art
Generally, to introduce a compound to a solid substrate, a method as illustrated in FIG. 1 is widely used. According to this method, on a solid substrate 1, there is formed a self-assembled thin film made of a silane compound or a thiol compound, an end of which has a functional group 2a for a linkage with the solid substrate 1 and the other end has a functional group 2c for a linkage with a compound to be introduced to the solid substrate 1. In the formation of the self-assembled thin film, molecular bodies, which are represented by a reference numeral 2b in FIG. 1, serve to increase the density of the thin film formed by molecular interaction therebetween. When the molecular bodies 2b are linear alkyl groups which are the simplest molecular structures, intermolecular spacing in a thin film formed therefrom is known to be about 0.5 nm.
As is well known, when a silane compound or a thiol compound with an additional end functional group is formed as a thin film on a specific solid substrate, chemical species, such as biomaterials, polymers, or nanoparticles, including DNAs and proteins, which are difficult to be immobilized on a solid substrate when used alone, can be easily and uniformly immobilized on the solid substrate. Therefore, many recent studies have been focused on immobilization of target compounds on solid substrates using such a silane compound or thiol compound.
Among these studies, in particular, a biochip technology for detection and assay of biomaterials, including DNAs and proteins, has been actively studied because it allows for the quick analysis of an interaction between a large number of biomaterials. In this regard, developments of techniques for the preparation of biochips with excellent sensitivity and selectivity have been competitively carried out.
A biochip includes a solid substrate 1 (a support layer) such as a silicon substrate or a glass substrate, a molecule layer (a linkage layer 2) formed on the solid substrate and having an end functional group that can be chemically bonded with DNA or protein, and a biomaterial layer (a detection layer 3) having DNA (complementary DNA) or protein that can selectively interact with a target material to be assayed, as shown in FIG. 2. Such a biochip is generally prepared as a pattern shape, like 1 of FIG. 3, and is then subjected to serial processes 7 and 8 to detect a target material to be assayed, which is well known in ordinary persons skilled in the art. In FIG. 3 is a solid substrate, 2 is a linkage layer, 3 is a detection layer (DNA single strand), 5 is a hybridized DNA double strand, 7 is sample insertion and DNA hybridization, and 8 is detection.
As shown in FIG. 4, when biomaterials, such as DNAs or proteins, which are immobilized on the solid substrate 1, selectively interact with specific target materials, they may be changed in structure or volume, in particular, toward a specific three-dimensional structure, which is well known in the pertinent art. In particular, in a case where biomaterials immobilized on the solid substrate 1 of the biochip are DNAs, a maximal cross-sectional diameter of DNA single strands immobilized on the detection layer 3 is about 1 nm or less. However, when the DNA single strands are hybridized with target DNA strands present in a sample to form double strand structures, a maximal cross-sectional diameter of the double strand structures is about 2.2 nm, which is larger than the maximal cross-sectional diameter of the DNA single strands. In FIG. 4 is a solid substrate, 2 is a linkage layer, 3 is a detection layer (DNA single strand), 5 is a hybridized DNA double helix, 7 is sample insertion and DNA hybridization.
It has been known that currently available DNA chip preparation technology has a limitation in presetting the spacing between DNA single strands used as the detection layer considering a size change that may result from hybridization with target DNA strands. If the DNA single strands are excessively densely spaced, the target DNA strands present in a sample cannot enter the detection layer due to a steric hindrance, which renders formation of double strand structures by hybridization with the DNA single strands immobilized on a solid substrate difficult. On the other hand, excessively sparse spacing of the DNA single strands on the solid substrate may lower the sensitivity of a DNA chip.
Hitherto, although a DNA chip has been mainly illustrated, there may arise similar problems to the above in a biochip except a DNA chip. For the forgoing reasons, an increase of the concentration of biomaterials constituting a detection layer per unit area alone cannot prepare a biochip with excellent selectivity and sensitivity. Rather, it is necessary to optimally space biomaterials of a detection layer to ensure constant sensitivity and selective interaction.
DNA double strands obtained by hybridization between target DNA strands and DNA strands immobilized on a solid substrate have a cross-sectional diameter that is about twice larger than the DNA strands immobilized on the solid substrate. In this regard, if DNA strands are excessively densely immobilized on a solid substrate of a DNA chip, steric hindrance may occur during subsequent hybridization with target DNA strands, thereby decreasing the efficiency of the DNA chip, as shown in (a) of FIG. 5 below. Therefore, to prepare a DNA chip with excellent selectivity and sensitivity, when DNA strands are immobilized on a solid substrate 1 to form a detection layer 3, the DNA strands must have uniform and close spacing considering a change in structure or volume that may be caused in subsequent hybridization, as shown in (b) of FIG. 5. In FIG. 5 is a solid substrate, 2 is a linkage layer, 3 is a detection layer (DNA single strand), and 6 is a target DNA strand.
The above-described steric hindrance problem that may be caused due to the structural change of biomaterials immobilized on a solid substrate during detection may also occur in a protein chip in which the sizes of proteins immobilized on a solid substrate are not large enough compared to those of target molecules (counterpart molecules selectively bonded with the proteins immobilized on the solid substrate), in addition to a DNA chip. For example, this may also be caused in a case where macromolecules, such as avidin, are bonded to micromolecules, such as biotin, immobilized on a solid substrate, which is widely used in a protein chip. Furthermore, the steric hindrance problem may also be caused at all chips in which chemical materials immobilized on a solid substrate are bonded to target materials, in addition to a biochip such as a DNA chip and a protein chip.
The best solution for this problem is to appropriately adjust the density of a linkage layer formed on a solid substrate. By doing so, the spacing between DNAs or proteins within a detection layer formed on the linkage layer can be adjusted.
Generally, the density of the linkage layer can be adjusted by controlling the concentration of a linkage layer material and the duration for linkage layer formation. Tarlov et al. reported a study in which adjustment of the concentration of a self-assembled thin film (corresponding to a linkage layer) material enables a change in spacing between chemical species [J. Am. Chem. Soc. 1998, 120, 9787]. According to this method, however, it is impossible to form a linkage layer with a uniform density distribution, which makes it difficult to control the spacing between DNAs formed on the linkage layer to a desired level. Recently, Georgiadis et al. reported a quantitative study measuring the degree of hybridization of DNAs after adjusting the spacing between chemical species by the same method as Tarlov et al. method. However, the above-described problem still remained [J. Am. Chem. Soc. 2002, 124, 14601].
Recently, Jun-Won Park and co-workers reported a method for adjusting the spacing between compounds used for a detection layer using conical dendron molecules [Korean Patent Laid-Open Publication No. 2002-0019325; Langmuir 19, 2003, p. 2357]. According to this method, when an aminosilane-modified solid substrate is treated with the dendron molecules, the lower end of each of which has 10 carboxyl groups and the upper end has a single amine group, a hydrogen bond between amine groups of the surface of the solid substrate and carboxyl groups of the lower ends of the dendron molecules occurs. Therefore, the dendron molecules are immobilized on the solid substrate and the amine groups present on the upper ends of the dendron molecules are spaced correspondingly to the maximal cross-sectional diameter of the dendron molecules. However, due to structural fragility, the dendron molecules may be easily changed structurally, such as bending or folding by rotation of a single bond, thereby reducing the spacing between the amine groups. Furthermore, overlapping of the dendron molecules may reduce the spacing between the amine groups. In this regard, this method cannot completely remove disadvantages caused by conventional methods. In addition, since the dendron molecules used in this method have a large number of functional groups, nonspecific binding occurrence which inhibits the performance of a DNA chip or a protein chip may increase.